The overall objective of the proposed research is to evaluate the gene regulatory pathway of the pneumococcal type 1 pilus and the interaction between the pilus and the innate and acquired immune responses. The type 1 pilus of S. pneumoniae is encoded by a small number of genes present in a subset of clinical strains. In the course of studies on the role of the type I pilus in virulence, the Malley and Dove laboratories jointly discovered that the genes encoding this pilus are bistably expressed, that the structural pilus protein RrgA acts as a negative regulator of pilus expression by inhibiting the positive regulator RlrA through a direct protein-protein interaction and that the pneumococcal type I pilus is controlled by a feedback- mediated bistable switch comprised of RlrA. Additionally, we have shown that RrgA is a potent activator of the innate immune receptor Toll-like receptor 2. Most recently, in trying to dissect the complex regulatory pathways of pilus gene expression, we uncovered a role for temperature in the control of type I pilus gene expression. We have also found that this silencing of pilus gene expression is dependent upon SP_1523, a protein that belongs to the HepA/RapA family. Our findings represent the first demonstration of a role of this conserved family of proteins in gene regulation. The complexity of the regulatory mechanisms of pilus expression and the interaction with an immune receptor raise the possibility that an intricate relationship between the bacterial structure and the host modulates the expression of the pilus. Here we propose to evaluate these issues further, by a combination of various approaches, including a thorough evaluation of how type 1 pilus genes are regulated by SP_1523 (Aim 1), and an in vivo analysis of the interaction between SP_1523, pilus expression, innate and acquired immune responses, using mutant, reporter and constitutively-expressing strains (Aim 2). Overall, these investigations will provide important insights into the mechanisms of gene regulation that may be applicable not only to S. pneumoniae but also to other bacteria, and also elucidate how the immune response to such tightly regulated genes may modify the selective advantages in colonization.